Voltage-gated ion channels are involved in nerve impulse propagation, synaptic transmission, muscle action potentials, and excitation/contraction coupling. Abnormal function of voltage-gated channels has been implicated in many neurological diseases, for example, epilepsy, episodic ataxia, and periodic paralyses. An understanding of the normal and pathological function of these channels could lead to the development of treatments for a number of neurological diseases. Voltage-activated ion channels are activated by changes in the voltage across the cell membrane. The classic voltage-activated potassium channels open when the membrane is depolarized. This has been shown to be due to the outward movement of an intrinsic voltage sensor, S4, which triggers the conformational changes that open the channel. However, how the movement of S4 is coupled to the opening of the channel is not understood. Recently, a new class of voltage-activated ion channels was cloned: hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels). These channels also contain a putative voltage sensor, S4. Surprisingly, the HCN channels open with the opposite polarity from the voltage-activated potassium channels; that is, the HCN channels open when the membrane is hyperpolarized. We hypothesize that S4 is also the voltage sensor in the HCN channels, but that the coupling between S4 movement and the opening of the channel involves a different mechanism than in voltage-activated potassium channels. The aim of the proposed project is to determine whether S4 is the voltage sensor in the HCN channels and to compare and contrast the S4 movement in these channels with the S4 movement in voltage-activated potassium channels. The movement of S4 will be measured in cysteine-substituted channels using cysteine-specific fluorescent probes or membrane-impermeable cysteine reagents. These measurements will be used to determine how S4 moves in the different channels and will further our understanding of the different mechanisms of opening of these two classes of channels.